Method for producing a recombinant protein using pollen

ABSTRACT

The invention relates to method for producing a recombinant protein using pollen. More specifically, the invention relates to method for producing a recombinant protein by introducing a recombinant gene into pollen using  Agrobacterium tumefaciens  via vacuum infiltration and then inducing pollen tube growth, and it makes possible to scale-up recombinant protein within short period of time and low production costs.

TECHNICAL FIELD

The present invention relates to a method for producing recombinantprotein using pollen.

More particularly, the present invention relates to a method forproducing recombinant protein coding a target gene by introducing thetarget gene into plant pollen by following transformation.

BACKGROUND ART

On attempts to produce recombinant protein in transgenic plant systems,much research has been conducted on the production of protein throughintracellular accumulation (cytoplasm or intracellular organelle) inspecific tissue or organ (seed, tuber, etc.) by employing plant, plantorgans, cultured plant cells or secretion systems (intracellular gap,secretion in medium). (Molony, Biotechnol. Eng., 9, 3, 1995; Kusnadi etal., Biotechnol. Bioeng., 56, 473, 1997; Smith & Glick, Biotechnol.Adv., 18, 85, 2000; Sijmons et al., Bio/Technology, 8, 217, 1990; Doran,Curr. Opin. Biotechnol., 11, 199, 2000; Boothe et al., Drug Develop.Res., 42, 171, 1997; Giddings et al., Nature Biotechnol., 18, 1151,2000).

Recombinant protein synthesizing systems that employ plants provide manyadvantages over other recombinant protein synthesizing systems employinganimal cells or bacteria, such as the moderate cost of cultivationcontrol, feasibility of scale-up, similarity to human glycosylation orfolding, absence of product impurities from transgenic animals orbacteria, and absence of contaminations of human-derived proteins.

Recombinant protein production systems that employ transgenic plant celllines are targeted to produce valuable proteins within a short period inbioreactors. This requires careful control and analysis techniques tooperate the bioreactors for the maintenance and proliferation oftransgenic plant cell lines.

Regarding an invention that employs plant pollen, U.S. Pat. No.5,929,300 describes a method for producing transgenic plants byintroducing a gene into the plant pollen using Agrobacterium,pollinating with the pistil, obtaining the seeds, and then germinatingthem to obtain the resulting transformed plant. However, it encounteredproblems with the long expression period of the target gene fromobtaining the seeds from the transformation and inbreeding and becauseof the complicated process. In addition, only the pollen of annualplants, including tobacco and cotton, could be used, except for thepollen of trees such as fine, gingko tree, etc.

In studying to solve the said problems, the inventor demonstrated thatwhen recombinant gene is introduced into plant pollen via Agrobacteriumtumefaciens and vacuum infiltration and then pollen tube growth isinduced, it could result in the mass production of recombinant proteinwithin a short period.

Up to now, there are no cases that employ plant pollen as the host forproducing recombinant protein. However, the following merits can beexpected when pollen is used to produce recombinant protein:

First, a production system with pollen cells can be utilized at a lowerprice compared to production systems with cell lines because pollencells can be collected plentifully at lower prices and there are nocomplicated costs from cell proliferation, maintenance of cellulargenetic stability and operation of bioreactors.

Second, the gene can be easily introduced via the method of particlebombardment, vacuum infiltration and electroporation in a productionsystem employing pollen cells (Tjokrokusumo et al., Plant Cell reports,19, 792, 2000; Fernando et al., Plant Cell reports, 10, 224, 2000).

Third, recombinant protein can be directly produced from pollen growthculture from a few times to a maximum of several days in a simple liquidmedium supplemented with 3˜4 kinds of chemicals, including sugar.

Fourth, plant pollen is a very excellent protein-producing host withtimely and economical efficiency, since the recombinant protein can becollected in a simple medium according to the use of secretion signalsof various kinds of protein secreted by the pollen.

The inventor accomplished the present invention through geneticintroduction and expression by recognizing that plant pollen is aproducing host of recombinant protein that can produce protein in ashort period at lower prices. Therefore, the object of the presentinvention is to provide a method for producing recombinant protein usingplant pollen as a producing host of various kinds of recombinant proteinfor use in testing, diagnosis and prevention and for industrial use.

DISCLOSURE OF THE INVENTION

According to the present invention, the object is achieved by collectingplant pollen, culturing them, introducing a target gene into the pollenvia vacuum infiltration, and expressing and identifying recombinantprotein from the germinated pollen.

The process of the present invention consists of isolating a pollen fromthe plant, culturing the pollen in a pollen growth medium, inserting atarget gene into an Agrobacterium vector and then transforming theAgrobacterium, infecting the transformed Agrobacterium into the culturedplant pollen, and growing the pollen with the target gene to obtain theresulting recombinant protein.

The present invention's method of producing recombinant protein usingpollen will be explained in greater detail below:

Step 1: Culturing Plant Pollen

Dehisced anthers were detached from lily flowers (Lilium longiflorum) orpine trees and their pollen grains were collected and then stored in adeep freezer at −70° C. until use. 0.5˜5 g of the pollen grains weresuspended in 200 mL of PGM and incubated at 25˜30° C. for 2˜24 hours inthe dark.

In the present invention, plant pollen is not limited to specific plantpollens, but all kinds of plant pollen can be used, including Pinaceae,Ginkgoaceae, as well as annual plants such as lily, cotton and tobacco.

In the present invention, the pollen growth medium (PGM) was preferablycomposed of 5˜10% sucrose, 0.5˜3 mM Ca(NO₃)₂, 50˜300 μM H₃BO₃, 0.001˜5mM KNO₃ and 0.1˜10 mM KH₂PO₄, more preferably 7% sucrose, 1.27 mMCa(NO₃)₂, 162 μM H₃BO₃, 0.99 mM KNO₃ and 3 mM KH₂PO₄.

Step 2: Construction of Recombinant Plasmid

A recombinant plasmid was constructed from the target gene by insertingto an Agrobacterium vector.

According to the present invention, ureB gene from Helicobacter pyloriand tPA gene from humans were used for the pollen transformation.

In order to produce protein encoded by ureB gene in accordance with thepresent invention, ureB gene was inserted into pBI121 with GUS reportergene to obtain pBIUreB, which is the recombinant plasmid forAgrobacterium transformation. To produce protein encoded by tPA (tissueplasminogen activator), the tPA gene was inserted into pBI121 with GUSreporter gene to obtain pBI/tPA or pSK/tPA, which is the recombinantplasmid for Agrobacterium transformation.

The recombinant proteins produced by the method of the present inventioncomprise some proteins produced from plant pollen, including ureB andtPA proteins.

Step 3: Transformation

The recombinant plasmid prepared in step 2 was introduced toAgrobacterium tumefaciens using the freeze-thaw method, incubated andselected in LB media with antibiotics such as kanamycin or cefotaxime.

Step 4: Introduction of Gene Via Vacuum Infiltration

In accordance with the present invention, the introduction of the geneto the plant pollen using Agrobacterium was carried out via particlebombardment, vacuum infiltration and electroporation.

Step 5: Identifying the Target Gene

Genomic DNA was extracted from germinated pollens with the recombinantgene according to the method of CTAB (Draper J. et al., Plant genetictransformation and gene expression: A laboratory manual, p204-208,Blackwell Scientific Publication, 1988).

The PCR technique can be used to determine whether the target gene wascorrectly introduced. In addition, the expression of the gene can beconfirmed by conventional molecular techniques such as Southernblotting, Northern blotting and Western blotting. Through the moleculartechniques, the enzymatic activity of β-glucronidase expressed frompBI121, as well as the expression of the gene, could be assayed byhistochemical staining, which turns into a blue color by reacting with asubstrate, X-gluc.

In accordance with the present invention, 10 hours to 7 days is requiredto express recombinant protein by transformation and grow the pollenwith the target gene.

The present invention will be explained in greater detail throughreference with the following examples. However, the following examplesare provided only to illustrate the present invention, and it should beunderstood that the scope of the present invention is not limitedthereto.

BRIEF DESCRIPTION OF THE INVENTION

The objective, features and other advantages of the present inventionwill be more clearly understood from the following detailed descriptiontaken in conjunction with the accompanying drawings, in which:

FIG. 1 is a microscopic photograph showing elongation of lily pollentubes

FIG. 2 shows maps of pBIUreB with ureB and pBI121 with GUS reporter gene

FIG. 3 shows the result of identifying ureB gene in pollen using the PCRtechnique

FIG. 4 is a microscopic photograph showing expression of GUS enzyme inpollen

FIG. 5 is a photograph showing expression of ureB mRNA in pollen

FIG. 6 shows expression of UreB protein in pollen

FIG. 7 is a photograph showing expression of tPA cDNA in E. coli treatedwith IPTG by SDS-PAGE

FIG. 8 shows kanamycin toxicity to growth of lily pollen and the resultof histochemical staining

FIG. 9 shows a result of Southern hybridization with genomic DNA ofpollen

FIG. 10 shows a result of Western blotting of pollen protein

BEST MODE FOR CARRYING OUT THE INVENTION EXAMPLE 1 Expression of ureBGene Using Lily Pollen

To obtain the pollen for the present invention, dehisced anthers weredetached from lily flowers (Lilium longiflorum) and their pollen grainswere collected and then stored in a deep freezer at −70° C. until use. 1g of the pollen grains were suspended in 200 mL of PGM composed of 7%sucrose, 1.27 mM Ca(NO₃)₂, 162 μM H₃BO₃, 0.99 mM KNO₃ and 3 mM KH₂PO₄and incubated at 27° C. for 3 hours in the dark. The result of theelongation of lily pollen according to times in growth medium isrepresented in FIG. 1.

To observe the expression of the gene in pollen, two kinds ofrecombinant plasmid were prepared. One of them was pBI121 with GUSreporter gene (Clontech) and the other was pBIUreB harboring ureB genefrom Helicobacter pylori (Genbank accession number AF352376: SEQ. No.3). It has also been reported that recombinant UreB protein has anefficacy as anti-cancer vaccine. Processes for constructing pBIUreB areas following:

Primer No. 1: 5′-ATC CTA GAA TGA AAA AGA TTA GCA-3′(SEQ. No. 1) andprimer No. 2: 5′-GAG CTC CTA GAA AAT GCT AAA GAG-3′(SEQ. No. 2) weresynthesized. From pH808 harboring urease gene from Helicobacter pylori(Lee et al., J. Biochem. Mol. 31, 240, 1998), 1.7 kb ureB DNA fragment(SEQ. No. 5) was amplified by PCR. The ureB DNA fragment was fused topT7 Blue T-vector (Novagene) to construct pTUreB. The resulting 1.7 kbureB DNA obtained from pTUreB was excised by cutting with Xba I and SacI was inserted to GUS-DNA deleted pBI121 by cutting with Xba I and Sac Ito obtain pBIUreB. The recombinant diagram was represented in FIG. 2.

To introduce the target gene via Agrobacterium transformation and vacuuminfiltration, the plasmids pBI121 and pBIUreB were transformed intoAgrobacterium tumefaciens A136(ATCC 51350) using the freeze-thaw method,respectively. Following this, they were selected and incubated in LBmedia with kanamycin.

To introduce the gene, the transformed Agrobacterium tumefaciensharboring pBI121 and pBIUreB, respectively, were mixed with pollenpre-incubated for 3 hours, and then vacuum infiltration was carried out.The transformed Agrobacterium was grown in LB liquid medium with 50 mg/Lof kanamycin and streptomycin, respectively. The bacterial cells werecollected by centrifuging 1 mL of the culture solution at 10,000×g for 3min. and suspended in the pollen growth medium, including 50 mg/mL ofpollen. The suspended solution was placed in a vacuum container undervacuum condition (−80 Pa, for 20 min) and filtered by filter paper. Thepollens were washed twice with PGM with 200 mg/L of cefotaxime. Thecollected pollen were grown in 10 mL of PGM at 27° C. for 20 hours.

EXPERIMENTAL EXAMPLE 1 Identifying Target Gene

To identify the target gene, fully-grown pollen tubes were collected.Genomic DNA was extracted from the pollen and PCR was performed for 35cycles of the following reaction using the genomic DNA as a template;94° C. 40 sec, 65° C. 1 min. 30 sec, 72° C. 2 min. Primers of SEQ. No. 1and SEQ. No. 2 were used to identify ureB DNA. The resulting DNA wasidentified in 1% agarose gel electrolysis.

As shown in FIG. 3, the 1.7 kb DNA fragment from the genomic DNA of thetransformed lily pollen was amplified and is not shown in theuntransformed pollen and in pBI121 transformed pollen.

EXPERIMENTAL EXAMPLE 2 Assay of Enzymatic Activity of GUS in Pollen

To confirm the expression of the target gene from the transformed pollenvia vacuum infiltration, the enzymatic activity of β-glucronidaseexpressed from pBI121 was measured by histochemical staining. In moredetail, the fully-grown pollen were suspended in X-gluc solution(100 μLof 100 mM NaPO₄, 50 μL of X-gluc(1 mg/mL of stock solution) and 850 μLof sterile water), placed for 3 hours in the dark until a blue colorcame into view and then observed under microscope. The results wererepresented in FIG. 4.

As shown in FIG. 4, the untransformed pollen showed pale blue and thetransformed pollen harboring pBI121 showed dark blue, which can confirmthe expression of GUS gene.

EXPERIMENTAL EXAMPLE 3 Expression of UreB Gene in Pollen by NorthernBlotting

To monitor the expression of ureB gene in the transformed pollenharboring pBIUreB, the production of ureB mRNA was investigated usingNorthern blotting. PTUreB was digested with Xba I and Sac I and theresulting 1.7 kb DNA fragment was used as a probe. Probe labeling wascarried out according to the manual of the random primed DNA labelingkit (Reche Molecular Biochemicals). RNA of fully-grown pollen tubes wasextracted in extraction buffer with guanidine isothiocyanate and waselectrophoresced on 1% formaldehyde gel. The gel was transferred to aNytran membrane (Schleicher & Schuell) and blotted with the probe.Hybridization was performed at 42° C. for 24 hours in a buffercontaining 5×SSC, 0.1% N-laurylsarcosin, 0.02% SDS, 2% blocking agentand 30% formamide, and washed with buffer containing 0.5×SSC and 0.5%SDS. After this, the results were monitored by X-ray autoradiography.The results are represented in FIG. 5.

As shown in FIG. 5, 1.7 kb ureB mRNA was not expressed in theuntransformed pollen, in pollen transformed by Agrobacterium and inpollen harboring pBI121, but was expressed in pollen harboring pBIUreB.

EXPERIMENTAL EXAMPLE 4 Expression of UreB Gene in Pollen by WesternBlotting

Pollen samples were ground using a mini-pestle and suspended in anextraction buffer (KPO₄ pH 7.6, 2 mM PMSF and 10 mM EDTA). The pollenlysates were centrifuged at 13,000×g for 10 min and the supernatant wascollected. According to Lowry's method, total protein was measuredquantitatively. 30 μg of protein was used for Western blotting assay.The extracted protein was electrophoresced on SDS-8% polyacrylamide geland the gel was transferred to Hybond-P-membrane (Amersham) in atransfer solution (0.025 M Tris-HCl, pH 8.3, 0.15 M glycine and 20% SDS)for 3˜4 hours at 300 mA followed with Western blotting. The primaryantibody used a rabbit anti-H. pylori urease IgG and the second antibodyused a goat anti-rabbit IgG HRP-conjugate. Results were monitored by theECL detection system (Amersham) and represented in FIG. 6. 67 kDa UreBprotein was used as a control of recombinant ureases from Helicobacterpylori.

As shown in FIG. 6, a band in the same position as that of 67 kDa UreBprotein of the control was not shown in untransformed pollen, in polleninfected by Agrobacterium and in pollen harboring pBI121, whereas UreBprotein was expressed successfully in pollen harboring pBIUreB.Meanwhile, 0.05% of UreB protein of the total soluble protein could beestimated through densitometric analysis in comparison to Westernblotting using the recombinant UreB protein from bacteria. The yield ofUreB protein was calculated in 50 μg UreB protein per 1 g of pollen.

EXAMPLE 2 Expression of tPA Gene Using Plant Pollen

rotein encoded by the

employing the lily pollen according to the present invention, tissueplasminogen activator (tPA) from human was expressed in transformedplant pollen.

According to the same method described in Example 1, lily pollen grainswere collected and suspended in PGM composed of 7% sucrose, 1.8 mMCa(NO₃)₂ and 1.6 mM H₃BO₃, and incubated at 27° C. for 16 hours in thedark.

For transformation, recombinant pBI/tPA and pSK/tPA constructs wereconstructed through the combination of pBI121 with tPA from human. Theprocess for making the constructs are as follows: the primer No. 3(sense) was designed to have Xba I restriction site fused to the startcodon of tPA nucleotide sequence, 5′-AATCTAGACATGGATGCAATGAAGA-3′ andthe primer No. 4 (antisense) was designed to contain the stop codonfollowed by SacI site, 5′-ATGATCTCTGGTCACGGTCGCATGTT-3′; FrompETPFR(ATCC #40403) harboring tPA gene, tPA was amplified using tPAspecific primers by PCR techniques. Polymerase Chain Reaction (PCR) wasperformed for 30 cycles of the following reaction: 94° C. 30 sec, 53° C.30 sec, 72° C. 2 min. The resulting 1.7 kb DNA fragment (SEQ. No. 6) wasligated to pT7BlueR (Novagen) plasmid to obtain the recombinant pT/tPA.From it, the tPA DNA was excised by cutting with XbaI and SacI to befused to pBluescript II SK (Stratagene) and to GUS-DNA deleted pBI121(Clonetech) by digesting with XbaI and SacI to obtain pSK/tPA andpBI/tPA (not shown). After this, two kinds of recombinant plasmid wereintroduced into E. coli DH5α and E. coli XL-1 Blue. And then, it wasidentified that the sequence inserted in the resulted 1.7 kb DNAfragment was tPA full length sequence.

Introduction of the recombinant plasmid pBI121 and pBI/tPA or pSK/tPAconstructs into Agrobacterium and induction of target gene via vacuuminfiltration were performed according to the method described inExample 1. Also, Agrobacterium tumefaciens LBA4404 transformed with therecombinant plasmid were grown in LB medium with kanamycin andcefotaxime. Concentration of kanamycin and cefotaxime and incubationcondition of the Agrobacterium tumefaciens LBA4404 transformed wascarried out in the same manner described in Example 1.

EXPERIMENTAL EXAMPLE 1 Expression of tPA in E. coli

To confirm whether the 1.7 kb DNA fragment from the recombinant plasmidconstructed in Example 2 encodes tPA full length, E coli XL-1BlueR cellstransformed with the pSK/tPA were grown to the early log phase(OD₆₀₀=0.5) in LB medium in shaker flasks and then treated with IPTG toa final concentration of 2 mM for further cultivation. The bacterialcells were collected by centrifugation at 12,000×g for 5 min at 4° C.and suspended in extraction buffer (50 mM Tris-Cl, pH 7.5, 5 mM EDTA,0.1% Tween 80, 2 mM PMSF) for disruption for three times in 2 min ofsonication on ice. From the cell lysates, the inclusion body wascollected by removing the supernatant following centrifugation at12,000×g for 20 min at 4° C., and then used for SDS-PAGE andimmuno-blotting. The LacZ-fused tPA was calculated to be a 66 kDaprotein (30 amino acids from the LacZ-α plus 562 amino acids of 62,900Da tPA protein), approximately. Human tPA standard was paralleled toshow its slightly larger size than the induced protein band. FIG. 7Arepresents a result of SDS-PAGE of inclusion bodies from IPTG-treated E.coli harboring pSK/tPA for 0˜5 hrs, wherein M represents protein sizemarker, C represents expression of tPA cDNA from pBluescript SK IItransformed. FIG. 7B represents a result of Western blotting of themembrane-transferred bacterial proteins.

As shown in FIG. 7, although not strong, the induced synthesis of the 66kDa protein evidently appeared at the one-hour point of the IPTGtreatment and continued to several hours examined in this experiment.From this result, the induced protein was almost expected to be thefused tPA, but its further confirmation was carried out by Westernblotting.

EXPERIMENTAL EXAMPLE 2 Assay of Enzymatic Activity of GUS in Pollen byHistochemical Staining

In order to demonstrate the expression of target gene from the pollen,activity of GUS enzyme was assayed by histochemical staining accordingto experimental example 1 of Example 1. FIG. 8A represents kanamycintoxicity to elongation of pollen tubes, wherein a, b, c, d, e and frepresent 0, 50, 100, 125, 150 and 200 mg/L of kanamycin added in thepollen growth media, respectively. FIG. 8B represents a result ofhistochemical staining of pollen tubes of the untransformed (con),pBI121 transformed (tra) and a magnified feature of the tra (mag).

As shown in FIG. 8, most of the transformed pollen grown with or withoutkanamycin was shown to be similarly demonstrating the GUS activity,implying that most of the pollen population successfully transformed byvacuum-infiltration.

EXPERIMENTAL EXAMPLE 3 Expression of tPA in Pollen by Southern Blotting

Fully-grown pollen tubes were washed several times with steriledistilled water, ground to a fine powder in the presence of liquidnitrogen using mortar and pestle, and used for extracting genomic DNAaccording to the CTAB/chloroform method (Taylor et al., 1993). GenomicDNA digested with XbaI and SacI was electrophoresced on a 0.8% agarosegel and transferred to a nylon membrane (Hybond N, Amersham) forhybridization with 1.7 kb tPA DNA. Probe labeling was done according tothe ECL random prime system (Pharmacia). Hybridization was performed at60° C. for 24 hrs in buffer containing 5×SSC, 0.1% SDS, 5% dextransulfate, 100 μg/mL denatured sheared salmon sperm DNA and 5% Pharmacialiquid block. Results were monitored by ECL detection system (Pharmacia)following anti-fluorescein-HRP reaction.

As shown in FIG. 7, 1.7 kb DNA fragment was evident from thetransformed, but none from the untransformed, strongly indicating tPADNA integration into the pollen chromosomal DNA. Total soluble proteinsestimated about 12% of the pollen weight were extracted for SDS-12% PAGEfollowed with Western blotting.

As shown in FIG. 9, protein bands in recognition to the tPA mAb could beobserved at the location nearly identical to the standard tPA andexpected to have a similar extent of glycosylation in mass in lilypollen. Approximately 0.05% of tPA protein of the total soluble proteinscould be estimated through densitometric analysis in comparison to thestandard tPA.

EXPERIMENTAL EXAMPLE 4 Expression of tPA in Pollen by Western Blotting

Expression of tPA recombinant protein was carried out by Westernblotting. Pollen samples were ground in liquid nitrogen using a mortarand pestle and suspended in extraction buffer (50 mM Tris-Cl, pH 7.5, 5mM EDTA, 0.1% Tween 80, 2 mM PMSF). The pollen lysates were centrifugedat 12,000×g for 15 min at 4° C. and the supernatant was used for SDS-12%polyacrylamide gel electrophoresis and Western blotting, according tothe standard technique (Sambrook and Russell, 2001). The tPA protein wasdetected using mAb-tPA (Biodesign, ME) and Western blue stabilizedsubstrate for alkaline phosphatase (Promega, WI). Single-chain tPA fromhuman melanoma cell culture (Sigma, Mo) was used as a standard. FIG. 10represents a result of Western blotting of pollen proteins, wherein Mrepresents the protein size marker, Con represents pBI121-transformedpollen, S30 and S60 represent the standard tPA 30 ng and 60 ng,respectively and Tra represents pBI/tPA transformed.

As shown in FIG. 10, the transferred 66 kDa protein bands onto PVDFmembrane could be detected as colored ones at their correspondinglocations in recognition to mAb against human tPA. Taken these resultstogether, the 1.7 kb PCR product amplified from pETPFR plasmid could beidentified for its encoded tPA protein in size and immune reaction.

INDUSTRIAL APPLICABILITY

As described before, the present invention comprises the use of plantpollen as a host for producing recombinant protein, which makes itpossible to easily produce the recombinant protein at lower prices incomparison to production systems employing animal cell lines due to theabsence of complicated costs in cell proliferation, maintenance ofcellular genetic stability and operation of bioreactor.

Also, the present invention can directly produce recombinant protein ina pollen growth culture from a few hours to maximum of several days in asimple liquid medium supplemented with 3˜4 kinds of chemicals, includingsugar. Therefore, the present invention is very useful in medical andgenetic engineering industries.

1. A method for producing recombinant protein using plant pollencomprising the steps of: isolating pollen from plant; culturing thepollen in a pollen growth medium; introducing a target gene intoAgrobacterium vector to be transformed; infecting the transformedAgrobacterium into the cultured plant pollen; and growing the pollenwith the gene in a pollen growth medium to obtain the resultingrecombinant protein.
 2. The method of production as set forth in claim1, wherein the plant pollen is isolated from Lilium longiflorum orPinaceae.
 3. The method of production as set forth in claim 1, whereinthe pollen growth medium is composed of 5˜10% sucrose, 0.5˜3 mMCa(NO₃)₂, 50˜300 μM H₂BO₃, 0.001˜5 mM KNO₃ and 0.1˜10 mM KH₂PO₄.
 4. Themethod of production as set forth in claim 1, wherein the gene isintroduced into Agrobacterium by a method selected from the groupconsisting of particle bombardment, vacuum infiltration andelectroporation.
 5. The method of production as set forth in claim 1,wherein the target gene is UreB gene from Helicobacter pylori and tissueplasminogen activator (tPA) from humans.